QuantiTect Probe PCR Kits enable sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C.

QuantiTect Probe PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Effective UNG digestion.

106 copies of two dUMP-containing PCR amplicons were treated with or without UNG and then amplified by real-time PCR. UNG was from various suppliers, and real-time PCR was performed using the master mix from the QuantiTect Probe PCR +UNG Kit. ΔCT on the Y-axis indicates CT values for non-UNG-treated samples subtracted from CT values for UNG-treated samples. The UNG supplied with the QuantiTect Kit provided the greatest ΔCT (9-12 cycles). This indicates that QIAGEN UNG digests carryover PCR products more effectively than UNG from other suppliers.

Two-step RT-PCR.

The QuantiTect Probe PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and cDNA template to the ready-to-use PCR master mix, and start the reaction on any real-time cycler (see also the table "Components of 2x QuantiTect Probe PCR Master Mix").

Highly specific amplification.

In comparison with other DNA polymerases, only HotStarTaq DNA Polymerase in combination with a unique buffer specifically amplified a 497 bp fragment (from 50 copies of an HIV-pol-gene construct in a background of 1 µg human genomic DNA). M: Markers.

Wide dynamic range in two-step PCR.

Duplicate reactions were performed on the Mx3005P using tenfold dilutions of human keukocyte cDNA (10 ng to 0.01 ng) and a TaqMan assay for IL1R2 (a cytokine). The QuantiTect Probe PCR Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 101%.

Wide dynamic range in real-time PCR.

Probe-based real-time PCR with UNG pretreatment was carried out using the QuantiTect Probe PCR +UNG Kit. Reactions were run in duplicate on the ABI PRISM 7900 using 10-fold dilutions of human leukocyte cDNA (100 ng to 10 pg) and a TaqMan assay for IL8 (a cytokine). The amplification plots were evenly spaced, leading to a high PCR efficiency of 94%.

High sensitivity and efficiency, and wide dynamic range.

Tenfold serial dilutions of leukocyte cDNA (100 ng to 1 pg) were analyzed in duplicate on the Mx3005 using the QuantiTect Probe PCR Kit with primers and a FAM-labeled probe specific for IL8 (interleukin 8). A PCR efficiency of 97% over 6 logs of template dilution was achieved, and as little as 1 pg of IL8 transcript was sensitively detected.

A major source of PCR contamination is the carryover of PCR products from previous reactions. Pretreatment with UNG prior to starting PCR ensures that any contaminating PCR products do not affect subsequent PCR. The combination of the specially optimized UNG solution and the proven PCR master mix in the QuantiTect Probe PCR +UNG Kit ensures effective elimination of carried-over PCR products together with reliable quantification of target sequences (see figures "Effective UNG digestion" and "Wide dynamic range in real-time PCR").

Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers

Procedure

QuantiTect Probe PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and DNA template to the ready-to-use PCR master mix, and start the reaction (see flowchart "Two-step RT-PCR"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions.

QuantiTect Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets or quantitative gDNA analysis on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Probe PCR Kit, which has been specially developed for fast cycling on these instruments.